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Has anyone tried using T7 or e.coli ligase instead of T4 ligase for ligating adapters? My lab uses custom adapters for amplicon sequencing on the MiSEQ. We have been having issues with adapter dimers and a strange "ladder" effect underneath our target band after barcoding PCR and after some investigation we think that it might be related to active T4 ligase in the PCR reaction. We use the 3' A-overhang of the PCR fragments to ligate on our adapters. Can you use T7 or other ligase types for TA ligations?? thanks for the help everyone!
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Ligation reaction should be cleaned up before PCR to reduce excess adapters, ligase and ligase buffer reagents carry over. Ligase is less likely cause of your observation because it would be deactivated very early in PCR temperatures and also it is active at low temperatures. T7 ligase also can be used as long as there are cohesive ends for ligation, but T4 is more robust than T7.
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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